Response of THP-1 cells to oxidative damage induced by AAPH.
Degree GrantorUniversity of Canterbury
Degree NameMaster of Science
Monocyte cells can be damaged when exposed to reactive oxidative species during inflammatory events within the body. This research project has examined in detail the cellular events associated with free radical damage induced death of monocyte cells, using the water soluble peroxyl radical generator 2, 2' -azobis(2-methylpropionamidine) dihydrochloride (AAPH) as a model of inflammation induced oxidative stress. The human monocyte-derived cell line THP-1 was incubated with 10mM AAPH in Earle's Balanced Salt Solution at 37°C for up to 24 hours. Protein hydroperoxide formation was observed to occur after a six hour lag, period which corresponded to the time in which most of the intracellular glutathione was lost from the cell. Cell viability loss, measured by the MTT reduction, was also observed after six hours of incubation while no caspase-3 activation was observed. A control ethanol treatment confirmed that caspase-3 could be activated within the THP-1 cells. Flow cytometry analysis failed to show phosphatidylserine exposure due to the AAPH treatment but DNA staining by propidium iodide confirmed the loss of viability seen with the MTT cell viability assay. This data strongly suggests that THP-1 cells undergo AAPH induced necrosis as a result of cellular damage including GSH loss and protein hydroperoxide formation. Microscopic examination of the cells identified many of the morphological characteristics of necrosis in the AAPH treated cells. Studies on the effect of AAPH on U937 cells were unsuccessful due to the possible presence of intracellular infection, resulting in an increased oxidant stress in the untreated cells.